Bio-rad Profinity eXact Purification Resin User Manual

For Technical Support, call your local Bio-Rad office, or in the U.S., call 1-800-4BIORAD (1-800-424-6723).Profinity eXact™ProteinPurification SystemI

recommended substitutes for NaCl or KCl. If using the 0.1 M sodium phosphate Profinity eXact bind/wash buffer, a higher sodium phosphateconcentration

Table 4. Chemical Compatibility (Partial List—See Appendix A forComplete List)RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceepptt

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonnRoche cOmplete Protease F–may lower yield s

Section 3Profinity eXact™System Strategies1. Basic purification strategy—create and purify an imprecise fusionthat ensures initial purification succes

Appendix A: N-terminal Target Protein Structure andCleavage KineticsAppendix B: Intrinsic Cleavage RatesAppendix C: Triggered Cleavage Ratesb. Determi

Section 4Cloning and Expression Process4.1 pPAL7 Expression VectorThe pPAL7 expression vector utilizes the T7lac promoter to stronglyexpress a Profin

Fig. 3. pPAL7 vector map.Table 5. Features of the pPAL7 Expression Vector.Vector Feature Vector PositionT7lacpromoter 1–17Profinity eXact tag 92–316

4.4 Cloning Region SequenceFig. 4. Sequence of Profinity eXact tag and cloning region.4.5 General Cloning GuidelinesThe pPAL7 vector is provided in

Optimal room-temperature purification of the target protein with theProfinity eXact resin is dependent upon the amino acid residues in the firsttwo po

Forward and reverse PCR primers starting with the predefined eight basesillustrated in Step 1 are used to PCR the target gene. Using the 3'Æ5&apo

Table of ContentsSection 1 Introduction...11.1 Introduction ...

Amount of purified PCR product used in reaction mix: 0.2–1 pmolT4 DNA polymerase: 5 U/pmol DNA fragment (Novagen; add DTT to 5 mM in reaction mix) or

Add 1–5 µl ligation mix to each 50 µl aliquot of competent cells.Incubate on ice, 30 min. Heat shock at 42°C, exactly 30 sec. Quicklyreturn to ice and

If the template is an ampicillin-resistant plasmid, it may be DpnI-treatedpost PCR amplification to ensure background-free transformants.2. Digest PCR

Add 1–5 µl ligation mix to each 50 µl singles aliquot of competentcells. Incubate on ice, 30 min. Heat shock at 42°C, exactly 30 sec.Quickly return to

If an imprecise fusion is desired, but the spacer length is not critical,the forward PCR primer may be designed for cloning into NcoI,BamHI, EcoRI, or

Add 1–5 µl ligation mix to each 50 µl aliquot of competent cells.Incubate on ice, 30 min. Heat shock at 42°C, exactly 30 sec. Quicklyreturn to ice and

3. Prepare overnight seed culture by inoculating 2 ml LB+100 µg/mlampicillin + 0.5% glucose.Shake culture at 37°C, 16–18 hours.4. Inoculate expression

The following procedure provides a quick screen for solubility and expression level:1. Pellet ~2 ml E. coliculture by centrifuging at 16,000 × g for 1

Fig. 6. Partioning profile. Precision Plus Protein™molecular weight markers were loaded inlane 1, followed by the total, soluble, and insoluble fracti

The Profinity eXact purification resin is fully functional at pH 6–8. Intrinsiccleavage of some proteins due to high OH–concentrations during thelysat

6.1.1Wash Buffer Selection...286.1.2Lysate Loading...296.1.3Prof

5.3.5 Profinity eXact MBP Control LysateThe Profinity eXact MBP control lysate is a lyophilized E. colilysate thatcontains recombinant Profinity eXac

Table 6. Recommended Lysis Volumes.CCuullttuurree CCeellll LLyyssiiss aanndd EExxttrraaccttiioonn VVoolluummee DDeennssiittyy RReeaaggeenntt

acetic acid to produce a chloride-free, Tris-acetate buffer with the proper pH.A Tris-phosphate buffer may likewise be made with Tris base and phospho

6.1.4 Cleavage and ElutionElution of target proteins is typically conducted by incubating the resin in 100 mM sodium fluoride, 100 mM sodium phosphat

1. Prechill bind/wash buffer on ice or at 4°C. 2. Remove storage buffer from packed spin column.Snap off end cap of spin column, and retain the cap fo

9. Centrifuge, repeat wash step.Remove remaining unbound proteins by centrifuging. Save the flowthrough from both wash steps and label fractions Wash

Part 1: Buffer Preparation 1. Prepare wash buffer (0.1 M sodium phosphate, pH 7.2).Avoid NaCl in the wash buffer. See Wash Buffer Selection section, p

10. Restart pumping at 3 ml/min (10 ml/min) of elution buffer and collect eluate containing purified target protein.The OD280may be used to monitor th

Be sure to prepare a 0.1 M phosphoric acid (cf. 0.1 N).4. Prepare storage buffer (0.02% sodium azide, 0.1 M sodium phosphate,pH 7.2).Part 2: Sample Lo

10. Incubate cartridge in elution buffer for at least 30 min, room temperature.If performing the incubation at 4°C, incubate the cartridge for at leas

Section 1IntroductionFig. 1. One-step tag cleavage and elution: Pure and simple.1.1 IntroductionAffinity tags are commonly used to facilitate the pu

4. Prepare storage buffer (0.02% sodium azide, 0.1 M sodium phosphate, pH 7.2).Part 2: Column Preparation5. Mix resin gently to completely resuspend 5

Part 5: Cleaning13. Wash column with at least 5 CV wash buffer.14. Clean column with 3 CV eleaning solution.15. Neutralize resin with 5 CV wash buffer

Fig. 7. N-terminal interactions of Profinity eXact affinity-tagged GB. The cognatesequence of the Profinity eXact tag is partially shown in yellow (KL

Although practical elution times are generally achieved with no spaceramino acids (+0 constructs), the use of a two amino acid spacer betweenthe Profi

The results show that while N-terminal structure can slow cleavage kinetics,the cleavage rate of the +2 construct is about five-times faster than the

Fig. 9. Effect of P1'–P2' amino acids on intrinsic cleavage rate (rates derived from agreen fluorescent protein (GFP) fusion in presence of

Fig. 10. Effect of P1'–P2' amino acids on triggered cleavage rate (rates derived from aGFP fusion in presence of 100 mM sodium fluoride, 100

Appendix DComprehensive Chemical CompatibilityListRReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraat

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonnDetergents Non-ionic (NP-40, Solubilizes p

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonnSulfhydrl b-mercaptoethanol 20 mM(Reducing

2release of highly purified recombinant protein with a native N-terminus.During elution, the cleaved Profinity eXact tag remains tightly associated wi

RReeaaggeenntt GGrroouupp RReeaaggeenntt CCoommmmeennttss AAcccceeppttaabbllee CCoonncceennttrraattiioonnImidazole 200 mMAmmonium sulfate 15%; use o

Elution • Incubate resin with elution buffer for longer time (tryup to 1 hour at room temperature or overnight at4°C)• If performing elution at 4°C, i

Fusion protein binds poorly to the resin: Fusion Protein • Proteins with high N-terminal structure may interferewith binding; adding amino acid spacer

7. Fang J and Ewald D, Expression cloned cDNA for 10-deacetylbaccatin III-10-O-acetyltransferase in Escherichia coli: a comparative study of threefusi

Appendix GOrdering InformationCatalog # Description156-3008 Profinity eXact Expression and Purification Starter Kit156-3000 Profinity eXact Cloning an

Appendix HLegal InformationProfinity eXact™vectors, tags and resins are exclusively licensed underpatent rights of Potomac Affinity Proteins. This pro

Commercial Entities Doing Business in the United StatesA separate license is required for any commercial use, including the use ofthese materials for

Life ScienceGroup00-0000 0000 Sig 1106Bulletin 0000 US/EG Rev ABio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 4BIORAD Austra

Section 2Product Description2.1 Profinity eXact™ System ComponentsTable 1. Profinity eXact System Components and Storage Conditions.CClloonniinngg

2.2 Profinity eXact Expression TechnologyFusion proteins with an N-terminal Profinity eXact tag are expressed withthe pPAL7 expression vector (5.9 kb

cleave at the C-terminus of a nine amino acid sequence (EEDKLFKAL).Through a third set of mutations, this cleavage reaction has been changedfrom a sel

2.4 Resin CharacteristicsThe resin attributes are summarized in Table 2.Table 2. Characteristics of Profinity eXact Purification Resin.Functional li